Abstract Expression of the major heat shock protein Hsp72 correlates with poor prognosis in many types of cancer, suggesting that Hsp72 can provide a selective advantage to cancer cells. However, our understanding of the distinct function of Hsp72 in cancer is very limited. The objective of this research program is to fill this gap of knowledge. Recently we established that Hsp72 in fact is essential for cancer cells, since depletion of Hsp72 prevented tumor formation in xenografts and precipitated growth inhibition and senescence in many cancer cell lines, but not in untransformed epithelial cells. Furthermore, we demonstrated that Hsp72 became essential for growth because of the permanent activation of PI3K or Ras oncogenic pathways in transformed cells. We hypothesize that elevated levels of Hsp72 seen in a variety of epithelial tumors function in preventing the endogenous oncogene-induced senescence program, thus allowing cancer cells to proliferate. Therefore, many tumors become addicted to high levels of Hsp72. The proposed project will elucidate mechanisms of the Hsp72- mediated control of the oncogene-induced senescence in cancer, using both cell culture, and animal models. A critical aspect of this work will be investigation the effects of NZ28, a novel inhibitor of the heat shock response developed by us, on tumors in animals. In Aim 1, we will establish the role of Hsp72 in the PI3K oncogenic pathway. We will test whether endogenous mPIK3CA oncogene determines dependence on Hsp72 of cancer lines established from human tumors. Further, we will elucidate signaling pathways controlled by Hsp72 that lead to the mPIK3CA-induced senescence. In Aim 2, we will establish the role of Hsp72 in neoplastic transformation by Her2 oncogene. Expression of Her2 oncogene in cells depleted of Hsp72 led to a paradoxical effect. Instead of transformation, these cells underwent growth arrest and senescence. Similar effect of Her2 was seen upon incubation of cells with an inhibitor of the heat shock response NZ28. Here, in both cell culture and animal experiments we will clarify how Hsp72 regulates Her2 signaling in tumors. We will investigate how Hsp72 knockout affects development of Her2-induced tumors in vivo. We will also establish whether the heat shock inhibitor NZ28 can cause tumor regression in a mouse model of Her2-positive breast tumor. Overall this program will clarify how Hsp72 controls tumor development.